Hepatitis C virus (“HCV”) was originally identified as a causative agent of transfusion-associated hepatitis that had a propensity to induce acute and chronic hepatitis and hepatocellular carcinoma. Choo et al., Science, 244, 359–362 (1989). It is a major cause of morbidity and mortality worldwide, considering that at least 50% of infected persons will develop chronic hepatitis, and 20% of these will further develop cirrhosis. Dienstag, Gastroenterology, 85, 439 (1983). No cure is currently available for treatment of chronic or acute HCV infection.
The complete nucleotide sequence and genetic organization of HCV has been fully elucidated by Choo et al., Proc. Natl. Acad. Sci. USA, 88, 2451–2455 (1991). The HCV genome of positive-stranded RNA consists of 9,379 nucleotides and has a single large open reading frame that could encode a viral polyprotein precursor of 3,011 amino acids. Although there is little overall similarity in sequence between that of HCV and other viruses whose sequence is known, a portion of the sequence (upstream of the 5′ end of the open reading frame) is similar to the analogously positioned sequence of pestiviral genomes. The polyprotein also displays significant sequence similarity to helicases encoded by animal pestiviruses and human flaviviruses, among others. Comparison of the hydrophobicity profiles of the sequence of encoded amino acids, and comparison of such a profile between HCV and a flavivirus (yellow fever virus), for example, has resulted in the assignment of regions of the HCV genome as relating to proteins forming the capsid or core (C), and the envelope (E1 and E2), as well as five regions that specify nonstructural proteins (NS1 through NS5).
The mechanisms whereby HCV causes acute hepatocellular injury and initiates the sequence of events leading to chronic liver disease and ultimately to hepatocellular carcinoma are not well understood. It is possible that both virus-related direct and immunologically-mediated indirect mechanisms play important roles in HCV chronic hepatitis. For example, a link between HCV infection and the presence of autoantibodies is well-established. Lenzi et al., Lancet, 338, 277–280 (1991). Unfortunately, analysis of the direct cytopathic effect of HCV for host liver cells has been hampered due to the lack of suitable animal models and tissue culture systems.
Several clinical observations support the hypothesis that the host immune response contributes to liver cell injury: first, infection acquired early in life occurring in an immunologically immature host leads to a chronic asymptomatic carrier state; second, chronic carriers without evidence of liver cell injury are common; and third, immunosuppression has a beneficial effect on liver cell injury in chronic hepatitis C. See Alter, in Viral Hepatitis And Liver Disease, (Hollinger et al., eds., 1991), 410–413. A recent report also demonstrated the presence of an HCV-specific, major histocompatibility complex (“HLA” or “MHC”) class I-restricted cytotoxic T cell (“CTL”) response in liver-infiltrating lymphocytes from two patients afflicted with chronic HCV hepatitis. Koziel et al., J. Immunol., 149, 3339–3344 (1992). More specifically, it is generally presumed that the                response to viral antigens is almost entirely T-cell dependent. Even the antibody response requires T-cell help. Thus susceptibility to virus infections is particularly associated with T cell dysfunction, though this tells us little about the effector mechanisms involved, since T cells are required both for antibody production and for some cytotoxic reactions.Roitt et al., Immunology (3d ed. 1993) at 15.3.        
Accordingly, central to the host immune response to attack by an intracellular agent (e.g., an infecting virus, bacterium, or other intracellular parasite) would be that which is mediated by the cellular immune system; in particular, by HLA class I CTL's. Class I antigens are cell surface glycoproteins that control the recognition by CTL's of modified (i.e., infected or otherwise altered, as in cancer) self cells, and of foreign cells. CTL-mediated lysis of virus infected host cells may lead to clearance of the virus or, if incomplete, such lysis may lead to viral persistence and eventually chronic tissue injury. Viral persistence and immunologically-mediated liver injury are thought to be important mechanisms leading to chronic hepatitis C after infection with HCV.
At its most fundamental level, the cellular immune response involves a multimolecular interaction between antigenic peptides, HLA molecules and T cell receptors (“TCR”) on the CTL. Unlike antigen recognition by B cell immunoglobulin receptors, the two general classes of T cells do not recognize native antigen in solution; rather, they recognize short antigenic peptides that have reached the cell surface via two quite different pathways (reviewed in Rothbard et al., Ann. Rev. Immunol., 9, 527–565 (1991); also, see Rötzschke et al., Immunol. Today, 12, 447–455 (1991)). The subject matter of the present invention centers on the induction of activity by one of these pathways, namely that involving the human CD8+ T cell and its counterpart in other mammalian species.
Human CD8+ T cells recognize short antigenic peptides (usually 9–11 residues in length) once presented to the antigen binding groove of HLA class I molecules. The antigen binding grooves, and, more generally, HLA class I molecules, are present at the surface of the cells in which each HLA class I molecule's precursor proteins were originally synthesized. As reported by Monaco (Immunol. Today, 13, 173–179 (1992)), such precursor proteins may be derived from an infecting virus. Accordingly, the antigenic peptides, processed within the CTL, are derived by proteolytic cleavage of endogenously synthesized antigen in the cytoplasm. The processed peptides are then bound by a family of transporter proteins (encoded within the HLA locus) that shuttle them into the lumen of the endoplasmic reticulum where they are scanned for the presence of HLA allele specific binding motifs by the antigen binding domain of resident HLA class I proteins. Peptides containing the appropriate motif are bound by the corresponding HLA class I molecule, which then associates with β2-microglobulin and moves to the cell surface as an integral membrane protein. At the cell surface, the integral membrane protein can present the antigenic peptide to the appropriately rearranged TCR on a CD8+ T cell. The T cell subset specificity of this interaction derives from the fact that the multimolecular HLA-peptide-TCR complex is stabilized by accessory interactions such as those between the CD8 molecule on the T cell and the HLA class I molecule involved in the complex.
At the present time, it is difficult to predict from the sequence of an antigenic protein how the protein will be processed and which peptide portions will bind HLA class I molecules and be presented to CTL's. Binding motifs have been predicted for some HLA class I molecules based on sequence analysis of peptides eluted from these molecules. Falk et al., Nature, 351, 290 (1991). However, not all peptides that match the motif will be recognized as CTL-recognizable epitopes. Moreover, even of the peptides that are processed and bind to HLA class I molecules, identifying which ones will contain CTL-recognizable epitopes is not yet predictable.
Due to work in other systems, it has been assumed that the HLA class I restricted, CD8+ CTL response to endogenously synthesized HCV antigens is responsible for the observed pathological consequences of chronic infection by this virus. Mondelli et al., Arch. Pathol. Lab. Med., 112, 489 (1988). This hypothesis was untestable until recently due to the absence of the necessary reagents and experimental systems. HCV has not been demonstrated to infect continuous human cell lines in tissue culture, and the only animal model of HCV (chimpanzee) infection that could be used for such studies involves a species for which the immune system is not sufficiently defined.
Irrespective of the mode of activity, it is evident that the CTL response with respect to HCV is deficient in cases of chronic HCV disease. Moreover, there are a large number of individuals who, having been infected with HCV, have since developed chronic HCV hepatitis. It would be desirable to stimulate the immune response in these individuals to respond to appropriate HCV antigens and thereby eliminate their infection. It would also be desirable to prevent the progression of an acute phase HCV infection to a chronic phase infection. Further, as there is no currently available vaccine for HCV infection of any sort, it would be desirable to establish such a vaccine, preferably based on a range of antigenic determinants. Accordingly, it is an object of the present invention to provide agents that strengthen or boost the cellular immune system to fight HCV hepatitis. It is a further object to provide pharmaceutical compositions that strengthen or boost the cellular immune system for fighting HCV hepatitis, both with reference to therapeutic and prophylactic uses.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.